A. Field of the Invention
The present invention relates generally to the fields of oncology, embryology, molecular biology and genetics. More specifically, the invention relates to the tumor biology of p53 mutants, molecules that interact with p53, and agents that are able to block p53-related defects in cellular differentiation.
B. Description of Related Art
Many mutant p53 alleles are oncogenic, and p53 was in fact first thought to be an oncogene as the first clones isolated corresponded to mutant forms expressed in immortalized cell lines. Expression of mutant p53 immortalizes primary fibroblasts, and in combination with mutant ras, transforms such cells (Jenkins et al., 1984; Parada et al., 1984; Eliyahu et al., 1985). Normal p53, however, functions as a tumor suppressor and its overexpression can inhibit the growth of various tumor cell lines and can block the transforming activity of a variety of oncogenes (Finlay et al., 1989; Donehower and Bradley, 1993).
p53 possesses several different biochemical activities, including transcriptional activation and repression, single-stranded DNA binding, and interaction with several heterologous proteins (Donehower and Bradley, 1993; Gottlieb and Oren, 1996; Haffner and Oren, 1995). These activities regulate a diverse array of biological processes, for example cell cycle transitions, apoptosis, and response to DNA damage (Donehower and Bradley, 1993; Gottlieb and Oren, 1996; Haffner and Oren, 1995). Elimination of p53 activity is thought to contribute to the initiation or progression of tumorigenesis by loss of cell cycle control, genomic instability, and the acquisition of novel properties, such as resistance to hypoxia (Donehower and Bradley, 1993; Gottlieb and Oren, 1996; Haffner and Oren, 1995; Graeber et al., 1996). Given this involvement in numerous fundamental processes, it is quite surprising that p53-null mice develop to term in the majority of cases (Donehower et al., 1992; Armstrong et al., 1995; Sah et al., 1995). However, p53 is expressed during early murine development in a pattern consistent with a role in differentiation (Schmid et al., 1991; Komarova et al., 1997), and the precise regulation of expression appears to be essential for normal development (Montes de Oca Luna et al., 1995; Jones et al., 1995).
Thus, although it is clear that p53 plays a key role in tumor development, and may be involved in differentiation as well, the particular mechanisms by which this molecule functions remain undefined. Moreover, it is unclear whether the role play by p53 in these two activities is related.
The present invention involves the role of p53 in the differentiation of embryonic tissues. More particularly, the present invention provides methods of the blocking of p53 function in embryonic tissues, and the use of these tissues as screening tools for substances that are capable of overcoming the p53-related block in differentiation, both in vitro and in vivo. These assays serve as a model for possible cancer therapeutics. In addition, methods for identifying additional cellular components that interact p53 or p53-related pathways are provided.
There is provided in the present invention a method of screening for agents that inhibit a p53-related block of embryonic cell differentiation comprising the steps of providing an undifferentiated embryonic cell; blocking the function of p53 in the cell such that the cell fails to differentiate; contacting the cell with a candidate agent; and comparing the differentiation of the contacted cell after the contacting with the differentiation of the cell in the absence of the candidate agent whereby an increase in differentiation indicates that the candidate agent is an inhibitor of the p53-related block of differentiation.
In particular embodiments, the method further comprises the step of comparing the differentiation of the cell after the contacting with the differentiation of the undifferentiated embryonic cell. In more particular embodiments, the cell may be an amphibian cell. In still more particular embodiments, amphibian cell is a Xenopus laevis cell. In other specific embodiments, the cell is an insect cell. In more particular embodiments, the insect cell is a Drosophila melanogaster cell. In further embodiments, it is contemplated that the cell may be a mammalian cell. In specific embodiments the cell may be a mouse cell, in alternate embodiments the mammalian cell is a human cell.
In particular aspects, the blocking is achieved by introducing into the cell a nucleic acid encoding a dominant negative mutant of p53. More specifically, the introducing may be achieved via electroporation, microinjection, particle bombardment, liposome transfer or viral infection. In certain aspects, the nucleic acid is a DNA; in other aspects, the nucleic acid is an RNA.
In certain specific embodiments, the blocking is achieved by introducing into the cell a nucleic acid encoding double minute-2 or a ortholog thereof. In other embodiments, the blocking is achieved by introducing into the cell an antisense nucleic acid for p53. In specific aspects, the candidate agent is a nucleic acid. In other embodiments, the candidate agent is polypeptide. In particular embodiments, the candidate agent is produced by a neighboring cell. In other embodiments, the neighboring cell is a second undifferentiated embryonic cell into which the candidate agent has been introduced.
In particular aspects, the differentiation is determined by culture of the undifferentiated embryonic cell in vitro under conditions where the cell differentiates.
More particularly the differentiation is determined by development of an embryo in vitro or in vivo.
Also provided is a method for identifying genes involved in p53-mediated embryonic cell differentiation comprising the steps of providing a plurality of undifferentiated Drosophila melanogaster embryonic cells; contacting the cells with a gene encoding a dominant negative mutant of p53 operably linked to a developmentally regulated promoter responsible for a given trait; mutagenizing the contacted cells; assessing development of the trait in the resulting flies; and determining the identity of a mutated cellular product in flies exhibiting trait development.
In certain embodiments, the mutagenizing may comprise radiation of the cells. In other embodiments, the mutagenizing may comprise contacting the cells with a DNA damaging agent. In still further embodiments, the mutagenizing comprises introducing mRNA into the cells.
In particular aspects of the present invention, the contacting comprises microinjection of a P-element comprising the mutant p53 gene and the promoter. In particular aspects, the promoter is the eyeless promoter and the trait is eye development.
In other aspects of the present invention, there is provided a method for identifying genes involved iri p53-mediated embryonic cell differentiation comprising the steps of providing a plurality of pluripotent embryonic stem cells transformed with a gene encoding a dominant negative mutant of p53 operably linked to an inducible promoter; contacting the cells with nucleic acid encoding a polypeptide and a factor that induces the inducible promoter; culturing the contacted cells under conditions permitting differentiation; and assessing the differentiation of the cells, wherein differentiation in a cell identifies a polypeptide involved in p53-mediated cell differentiation.
In preferred aspects, the method further comprises contacting the cells with a plurality of nucleic acids encoding polypeptides. In certain embodiments, the plurality of nucleic acids is an expression vector library.
In particular embodiments, the inducible promoter is the ecdysone response promoter and the factor is ecdysone. In alternate embodiments, the inducible promoter is the metallothionein promoter and the factor is a heavy metal. In yet another embodiment, the inducible promoter is the tetracycline promoter and the factor is tetracycline. In still another alternative embodiment, the inducible promoter is a heat shock promoter and the factor is heat.
In particular aspects, the conditions include culture without LIF. In preferred embodiments, the differentiation comprises formation of hematopoietic cells, macrophages and cardiac cells. In other embodiments, the conditions further include culture on bacterial grade plastic and the differentiation comprises formation of erythroid cells. In other embodiments the conditions further include culture with retinoic acid and the differentiation comprises formation of neural cells. In still another embodiments, the conditions further include culture with erythropoietin and the differentiation comprises formation of hematopoietic cells. In a further alternative embodiments, the conditions further include culture with interleukin 3 and the differentiation comprises formation of macrophages, neutrophils and mast cells. In yet another alternative, the conditions further include culture with interleukin 3, interleukin 1, and macrophage colony stimulating factor or granulocyte-macrophage colony stimulating factor and the differentiation comprises macrophages. In other embodiments, the conditions further include culture with bone morphogenetic protein 4 and the differentiation comprises hematopoietic cells.
In particular aspects, the the embryonic stem cells are mammalian cells. The mammalian cells may be mouse cells. In those embodiments, where the cells are mouse cells, the culturing may comprise injected the contacted cells into a mouse blastocyst and implanting the blastocyst in a foster mother. Alternatively, the mammalian cells are human cells. In other preferred embodiments, the embryonic stem cells are amphibian cells. In particular embodiments, the amphibian cells are Xenopus laevis cells. In an alternate preferred embodiments the embryonic stem cells are insect cells. More particularly the insect cells may be Drosophila melanogaster cells.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.